Controlling Ligand Binding in Myoglobin by Mutagenesis

Federica Draghi,Adriana Erica Miele,Carlo Travaglini-Allocatelli,Beatrice Vallone,Maurizio Brunori,Quentin H Gibson,John S Olson

doi:10.1074/jbc.m109206200

Abstract

A quadruple mutant of sperm whale myoglobin was constructed to mimic the structure found in Ascaris suum hemoglobin. The replacements include His(E7)-->Gln, Leu(B10)-->Tyr, Thr(E10)--> Arg, and Ile(G8)-->Phe. Single, double, and triple mutants were characterized to dissect out the effects of the individual substitutions. The crystal structures of the deoxy and oxy forms of the quadruple mutant were determined and compared with that of native Ascaris hemoglobin. Tyr(B10) myoglobin displays low O(2) affinity, high dissociation rate constants, and heterogeneous kinetic behavior, suggesting unfavorable steric interactions between the B10 phenol side chain and His(E7). In contrast, all mutants containing the Tyr(B10)/Gln(E7) pair show high O(2) affinity, low dissociation rate constants, and simple, monophasic kinetic behavior. Replacement of Ile(107) with Phe enhances nanosecond geminate recombination singly and in combination with the Tyr(B10)/Gln(E7)/Arg(E10) mutation by limiting access to the Xe4 site. These kinetic results and comparisons with native Ascaris hemoglobin demonstrate the importance of distal pocket cavities in governing the kinetics of ligand binding. The approximately 150-fold higher O(2) affinity of Ascaris hemoglobin compared with that for Tyr(B10)/Gln(E7)-containing myoglobin mutants appears to be the result of favorable proximal effects in the Ascaris protein, due to a staggered orientation of His(F8), the lack of a hydrogen bonding lattice between the F4, F7, and F8 residues, and the presence of a large polar Trp(G5) residue in the interior portion of the proximal heme pocket.

Highlights

More than 50 years ago, Davenport [1] showed that the ultrahigh affinity of Ascaris suum hemoglobin for O2 is due to a remarkably small rate constant for O2 dissociation (Յ0.01 sϪ1)
Rate constants for O2, CO, and NO binding were measured for Mb mutants containing all four Y, Q, R, and F replacements
Parameters for Ascaris Hb and some of the mutants were taken from Gibson and Smith [66], Springer et al [35], and Travaglini-Allocatelli et al [4]

Summary

Introduction

More than 50 years ago, Davenport [1] showed that the ultrahigh affinity of Ascaris suum hemoglobin for O2 is due to a remarkably small rate constant for O2 dissociation (Յ0.01 sϪ1). In 1994, De Baere et al [2] and Kloek et al [3] suggested that the structural cause of the low dissociation rate was electrostatic stabilization of bound O2 by two hydrogen bonds, a very strong one with Tyr(B10) and a weaker one with Gln(E7). These conclusions were based on the effects of Tyr(B10) 3 Phe and Gln(E7) 3 Leu mutations on the rate of O2 dissociaton from recombinant Ascaris heme domain I. The functional results are correlated with the deoxygenated and oxygenated crystal structures of YQRF myoglobin and those reported previously for A. suum hemoglobin [13]

Results

Discussion

Conclusion